Searching for virulence factors in the non-pathogenic parasite to humans Leishmania tarentolae

Searching for virulence factors in the non-pathogenic

parasite to humans Leishmania tarentolae

H. AZIZI1#, K. HASSANI1,2#, Y. TASLIMI1, H. SHATERI NAJAFABADI3,

B. PAPADOPOULOU4 and S. RAFATI1*

1Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran2Department of Biotechnology, University College of Science, University of Tehran, Tehran, Iran3 Institute of Parasitology, McGill University, Montreal, Canada4Research Centre in Infectious Diseases, CHUL Research Centre and Department of Medical Biology, Faculty of Medicine,Laval University, 2705 Laurier Blvd., Quebec (QC), Canada G1V 4G2

(Received 15 September 2008; revised 7 November 2008 and 8 January 2009; accepted 8 February 2009; first published online 6 May 2009)

SUMMARY

Leishmania protozoa are obligate intracellular parasites that reside in the phagolysosome of host macrophages and cause a

large spectrum of pathologies to humans known as leishmaniases. The outcome of the disease is highly dependent on the

parasite species and on its ascribed virulence factors and the immune status of the host. Characterization of the genome

composition of non-pathogenic species could ultimately open new horizons in Leishmania developmental biology and also

the disease monitoring. Here, we provide evidence that the lizard non-pathogenic to humans Leishmania tarentolae species

expresses anAmastin-like gene, cysteine protease B (CPB), lipophosphoglycan LPG3 and the leishmanolysinGP63, genes

well-known for their potential role in the parasite virulence. These genes were expressed at levels comparable to those in

L. major and L. infantum both at the level of mRNA and protein. Alignment of the L. tarentolae proteins with their

counterparts in the pathogenic species demonstrated that the degree of similarity varied from 59% and 60% for Amastin,

89% for LPG3 and 71% and 68% forCPB, in L. major and L. infantum, respectively. Interestingly, theA2 gene, expressed

specifically by the L. donovani complex which promotes visceralization, was absent in L. tarentolae. These findings suggest

that the lack of pathogenicity in L. tarentolae is not associated with known virulence genes such as LPG3, CPB,GP63 and

Amastin, and that other factors either unique to L. tarentolae or missing from this species may be responsible for the non-

pathogenic potential of this lizard parasite.

Key words: pathogenic, non-pathogenic, Leishmania major, Leishmania infantum, Leishmania tarentolae, virulence factors.

INTRODUCTION

Protozoan parasites of the Leishmania genus are

causative agents of a broad spectrum of diseases

generally termed as leishmaniases. Disease manifes-

tations range from cutaneous leishmaniasis caused by

Leishmania major, Leishmania aethiopica, Leishmania

tropica, Leishmania mexicana and Leishmania ama-

zonensis, to muco-cutaneous leishmaniasis caused by

Leishmania braziliensis and Leishmania guyanensis

and to potentially deadly visceral leishmaniasis or

Kala-azar caused by the Leishmania donovani com-

plex (L. donovani and Leishmania infantum) (Murray

et al. 2005). Leishmaniasis is associated with 2.4

million disability-adjusted life years and 70000 deaths

per year (Desjeux, 2004). Extracellular flagellated

promastigotes of Leishmania live and multiply in the

mid-gut of the female Phlebotomine sandfly. Pro-

mastigotes enter the skin of the vertebrate host by the

bite of the sandfly, are phagocytosed by immune

cells, and transform into non-motile ovoid-shaped

amastigotes that are able to propagate within the

harsh environment of the macrophage phagolyso-

some.

Not all members of Leishmania genus are parasites

of mammals. Certain species ofLeishmania including

Leishmania tarentolae are lizard parasites. Whether

these parasites should be included within the genus

Leishmania has been a matter of debate. However,

molecular phylogenetic studies have suggested that

lizard Leishmania, alternatively named Sauroleish-

mania, have evolved from Old World Leishmania

after separation of the New World Vianna subgenus

(Croan et al. 1997). L. tarentolae is a lizard parasite

strain, which has never been found associated with

any leishmaniasis in humans and is thus considered

as non-pathogenic to humans. L. tarentolae has

been used as a model organism for studying unique

features of Kinetoplastids such as RNA editing

(Landweber and Gilbert, 1994; Maslov et al.

1994). However, as Noyes et al. (1998) have also

* Corresponding author: Molecular Immunology andVaccine Research Laboratory, Pasteur Institute of Iran,Tehran, Iran. Tel: +98 21 66953311. Fax: +98 2166465132. E-mail : [email protected] or [email protected]# These authors contributed equally to this work.

723

Parasitology (2009), 136, 723–735. f Cambridge University Press 2009

doi:10.1017/S0031182009005873 Printed in the United Kingdom

indicated previously, comparative studies of lizard

Leishmania and virulent Leishmania can be of critical

importance towards understanding the pathogenicity

of Leishmania and its relationships with the host.

Leishmania spp. form complex host-parasite inter-

actions and are able to alter the host immune re-

sponse for their benefit. In order to explain the

absence of pathogenicity in L. tarentolae, we tested

whether known virulence factors of pathogenic

Leishmania species were present and or expressed in

L. tarentolae.

A number ofLeishmania’s virulence factors such as

GP63 (Joshi et al. 2002; McGwire et al. 2002; Yao

et al. 2003; Campbell et al. 1992) and cysteine pro-

teases (CPs) (Alexander et al. 1998; Rosenthal, 1999;

Mundodi et al. 2002; Mottram et al. 2004) have been

characterized and their roles have been extensively

studied; some others such asLPG3 (Descoteaux et al.

2002; Ouakad et al. 2007b), Amastins (Salotra et al.

2006) and A2 (Zhang andMatlashewski, 1997, 2001;

Zhang et al. 2003) have also been identified. GP63

(also called Major Surface Protease) is one of the

most extensively studied virulence factors of Leish-

mania. GP63 is a surface endopeptidase with a wide

range of substrate and pH specificity, well suited for

its dual expression in promastigote and amastigote

stages. Targeted gene deletion studies have shown a

crucial role for GP63 in the pathogenicity of Leish-

mania ; gp63nul mutants of L. major showed reduced

pathogenicity and were weakened in forming lesions

in BALB/C mice in comparison to wild types (Joshi

et al. 2002). As another group of virulence factors of

Leishmania, the cysteine proteases have been studied

mostly in L. mexicana due to their crucial role in its

pathogenicity. Most studies on cysteine proteases in

L. mexicana have been done on the most abundant

Cysteine Protease families A, B and C. Similar to

gp63x/x L. major, Dcpb L. mexicana have impaired

infectivity against BALB/c mice (Mottram et al.

1998; Alexander et al. 1998). Furthermore, Williams

et al. (2006) have shown that CPB and CPA are es-

sential for parasite autophagy during promastigote-

amastigote transformation. The dominant surface

molecule of promastigotes is lipophosphoglycan

(LPG). This molecule plays important roles in the

early stages of the infection, wherein the parasite is

still in the promastigote form, namely interaction

with the complement system, phagocytosis and pro-

tection of promastigotes against lysis in the phago-

lysosome (Spath et al. 2003). LPG3, a molecular

chaperone, is part of the metabolic pathway of

synthesis of LPG as well as phosphoglycan residues

that are added to extracellular proteins, such asGP63,

GP46, sAP and GPI-anchored proteins (Descoteaux

et al. 2002). The expression level of surface proteins

such as GP63 and GP46 is greatly reduced in lpg3nul

mutants (Descoteaux et al. 2002). Based on these

findings it was proposed that the role of LPG3 in

virulence should be exerted through its role in the

synthesis of LPG, GPI-anchored proteins (such as

GP63) and other phosphoglycan-bearing (PG)

molecules (Descoteaux et al. 2002). Amastins belong

to a large gene family of surface proteins that are

developmentally regulated in the amastigote stage of

Leishmania (Wu et al. 2000). Although the biological

function of amastins remains still unknown, it is

hypothesized that amastins may play a role in proton

or ion traffic across the membrane to adjust the

cytoplasmic pH under the harsh conditions of the

phagolysosome (Rochette et al. 2005). Like amastins,

A2 is an amastigote-specific protein, originally dis-

covered in L. donovani. The exact function of A2

protein in Leishmania is unknown. However, gene

silencing ofA2 inL. donovani dramatically decreased

its viability in mammalian macrophages and deterio-

rated its pathogenicity (Zhang and Matlashewski,

1997). Interestingly, over-expression of A2 in

L. major resulted in visceralization of the infection

(Zhang and Matlashewski, 1997; Garin et al. 2005).

In this study, we tested whether well-character-

ized virulence factors in the pathogenic Leishmania

species (e.g. L. major, L. infantum and L. braziliensis)

were present and or expressed in the non-pathogenic

L. tarentolae species in order to gain a better insight

into the factors responsible for the lack of patho-

genicity in L. tarentolae. We found that all the well-

known virulence factors that we tested here, with the

only exception of A2, were present and expressed in

L. tarentolae at ratios similar to those of the patho-

genic species.

MATERIALS AND METHODS

Preparation of stationary phase promastigotes

L. major (MRHO/IR75/ER), L. infantum (MCAN/

98/LLM-877) and L. tarentolae (ATCC 30267)

promastigotes were grown at 26 xC in complete

M199medium containing 10% heat-inactivated FCS,

40 mMHEPES, 0.1 mM adenosine, 0.5 mg/ml haemin

and 50 mg/ml gentamicin, and were allowed to mul-

tiply until they reached a density of 2–3r107 fol-

lowing 5 days of growth at 26 xC. The stationary

phase promastigotes were then utilized for macro-

phage cells (MQ) infection and RNA extraction.

Probe preparation

The ORF of cysteine protease type I (GenBank

Accession no. U43706) was digested using BamHI-

HindIII from vector pGEM-cpb (Rafati et al. 2001).

The ORF of GP63 from L. major (GenBank Ac-

cession no. AF039721) was digested usingXbaI from

a vector kindly provided by Dr Mahboudi (Pasteur

Institute of Iran). LPG3 ORF (GeneDB Accession

no. LmjF29.0760) was digested using EcoRI-SalI

from pGEM-lpg3 vector prepared previously in

our lab (unpublished). The A2 ORF (Accession no.

H. Azizi and others 724

S68693) was digested with BamHI from a vector

kindly provided by Dr Greg Matlashewski (McGill

University, Canada). Partial sequences from amas-

tins Lmj24.1270, Lmj28.1390, Lmj30.0870 were

digested by EcoRI from vectors described previously

(Rochette et al. 2005). Following digestion and ex-

traction of fragments from agarose gel, probes were

prepared by random primer extension using Klenow

enzyme (Roche). In certain sections of library

screening, amastin probes were prepared by a non-

radioactive Digoxigenin (DIG) labelling (Roche) in a

similar process.

Total DNA preparation and hybridizations

Total DNA of L. tarentolae (strain ATCC 30267),

L. major (strain MRHO/IR75/ER) and L. infantum

(MCAN/98/LLM-877) was prepared from log-

phase parasites using the standard proteinase K

(Roche) and phenol extraction method (Sambrook

and Russell, 2001) and digested using PstI (Roche).

Southern blotting was done according to standard

methods (Sambrook and Russell, 2001). Resulting

Nylon membranes (Roche) were hybridized to32P-labelled probes in 20 ml of Church buffer (0.5 M

phosphate-buffered saline (PBS) containing 1%

BSA, 1 mM EDTA and 7% SDS) at 65 xC overnight.

Non-stringent and stringent washes were done in

25 ml of NaPi buffer for 15 min at 25 xC and 65 xC,

respectively. When necessary, non-stringent wash-

ing at 42 xC was also performed for confirmation.

Radiofilms (Kodak) were exposed overnight or for 2

days for confirmation.

Library screening

One mg of the previously described cosmid library of

L. tarentolae (Genest et al. 2008) was transformed to

E. coli DH5a by chemical transformation. Colony

lifting was done according to standard methods

(Sambrook and Russell, 2001) and the resulting

nylon membranes were hybridized to the probe for

L. major’s amastin Lmj24.1270 under non-stringent

conditions as stated in the previous section. The

transformation and screening was performed 6 times

in order to achieve high genome coverage. Cosmids

that were hybridized to the amastin probe were

digested using PstI and re-blotted against the probe

and the hybridizing fragment was subcloned in

pGEM-2 vector (Promega) after dephosphorylation

(Shrimp Alkaline Phosphatase, Roche). The cloned

fragment was sequenced using universal T7 and SP6

primers.

Sequence analysis

Orthologues of the sequences acquired from

L. tarentolae were found in the genomes of L. major,

L. infantum and L. braziliensis using GeneDB

BLAST search tool (for each gene, the hit with

the lowest E-value was chosen). Pairwise sequence

alignments were performed on translations of the

acquired sequences at EBI website. Multiple se-

quence alignment was performed using web-based

CLUSTALW at EBI bioinformatics tools. Trans-

membrane helices of L. tarentolae’s amastin were

predicted using TMHMM server version 2 (Krogh

et al. 2001).

Amastigote preparation

Peritoneal exudate cells from naıve BALB/c mice

were used as a source of resident macrophages (MQ).

Briefly, the peritoneal cavity was washed with 5 ml

of cold RPMI 1640 (Sigma) medium, and then

the medium containing peritoneal macrophages was

recovered. Cells were cultured in RPMI medium

1640 supplemented with 5% FCS, 2 mM glutamine,

50 mM 2-mercaptoethanol, 10 mM HEPES and

40 mg/ml gentamicin at a density of 106 cells/well, and

incubated at 37 xC in 5% CO2 for 24 h. In order

to infect MQ, the stationary phase Leishmania pro-

mastigotes were added at the ratio of 10 : 1 (parasites

versus macrophages). The cultures were washed

after 4 h to exclude free parasites and then incubated

for 5 days. Infected attached MQs were collected

using cold PBS 1X (Breton et al. 2005).

RNA extraction

Stationary phase promastigotes and amastigotes of

L. tarentolae, L. major and L. infantum were utilized

for RNA purification using RNeasy Mini kit

(Qiagen). Briefly, after washing the promastigotes

twice with PBS, total RNA was extracted according

to the supplier kit. For total amastigote RNA iso-

lation, the infected murine peritoneal macrophages

were detached from the plate with cold PBS and

lysed according to QIAGEN RNAeasy kit. To pre-

vent co-purifying genomic DNA with RNA, on

column DNase treatment was done using RNase

free DNase set (Qiagen). The quality of the RNA

was assessed by spectrophotometric analysis using

NanoDrop ND-1000. Integrity of the RNA was

checked on 1% formaldehyde agarose gels. To ensure

that the obtained amastigote RNA is free of host

(macrophage) RNA, we looked for the presence of

murine ribosomal RNA on the gel.

cDNA synthesis

One mg of total RNA from each sample was reverse-

transcribed to cDNA using the Omniscript RT kit

(Qiagen) and random hexamer primers as described

by the supplier. After preparation of the mixture,

samples were incubated at 37 xC for 1 h, heat-

inactivated at 70 xC for 6 min and subsequently

stored in x20 xC.

Virulence factors in Leishmania tarentolae 725

Real-time PCR

cDNAs from all samples were utilized as a template

for the real-time polymerase chain reaction. Quanti-

fication of the mRNA corresponding to LPG3,

Amastin and CPB was done in the 7500 real-time

PCR system (Applied Biosystems) using QuantiFast

SYBR Green master mix from Qiagen. Amplifi-

cation of theAmastin fragment (185 bp) fromL.major

(Accession no. LmjF28.1400) and L. infantum (Ac-

cession no. LinJ28.1460) mRNA was carried out

using the same primers (AmastinF, 5k-GCGGTGG-

AGACGTGCTG-3k and AmastinR, 5k-GTGTAG-

GTAGAGTTGTCGCAGTTGT-3k). Amplification

of a 100 bp region from the L. tarentolae Amastin

transcript (GenBank Accession no. EU041954.1)

was accomplished using a primer pair specific to this

parasite (Amastin-tF, 5k-GCTTACCTGATGAT-

GGCGGATA-3k and Amastin-tR, 5k-CCAGTTG-

CGATGTGCAGATC-3k). The primers for LPG3

(LPG3F, 5k-TCTTCATCACGGACGAGTTCC-

3k and LPG3R, 5k-TTGCGCACAAGTTTCTT-

CTT-3k) were designed from a conserved region in

a multiple sequence alignment of L. major, L. in-

fantum, L. donovani and L. braziliensis LPG3

genes (Accession nos LmjF29.0760, LinJ29.0920,

AF369892 and LbrM29-V2.0780, respectively) and

were successfully used in amplifying the LPG3

transcript of L. tarentolae. The primers CPB-F,

5k-AACTTCGAGCGCAACCT-3k and CPB-R,

5k-GGTCAAAGAACTTCGTGATC-3k also servedfor amplifying an 85 bp fragment of L. infantum

(LinJ08.0980) and of L. major (LmjF08.1080).

Likewise, utilizing the primers CPB-LtF: 5k-AGC-

GGCGGGCTGATGT-3k and CPB-LtR: 5k-GAG-

CACTCGGGCGCATAG-3k, a 115 bp region of

L. tarentolae CPB (Accesion. no. EU860161) was

successfully obtained. The primers that served

to amplify a 100 bp rRNA region (LmjF32.3420

and LinJ32.3980 for L. major and L. infantum,

respectively) were rRNA-F: 5k-CGGTTTTTCC-

CTGATGTGGTAA-3k and rRNA-R: 5k-GGAA-

GCGTGGTCGACGTAGA-3k. Finally, GAPDH

from all species was amplified using the 5k-TCG-

CTCTCGTGGACATGAGCA-3k (forward) and

5k- CTTGAGCGACACGCCGTCCA-3k (reverse).PCR reactions were performed at 95 xC for 5 min

followed by 40 cycles of 95 xC for 15 s and 60 xC for

1 min. Melting curve analysis demonstrated specific

single PCR products for all reactions according to

the amplicon melting temperatures. Furthermore,

2 housekeeper genes, GAPDH and rRNA45 were

employed as internal controls for normalization. All

the primers were designed utilizing Primer Express

software v.2 (Applied Biosystems). Relative quantity

of all genes was determined through DDCT Method

(SDS software, Applied Biosystems). RT negative

controls were also performed to check for the pres-

ence of contaminated DNA.

SDS-PAGE and immunoblotting

Stationary phase promastigotes were collected from

in vitro cultures by centrifugation (10 min, 1500 g,

4 xC), washed in PBS and lysed in SDS-PAGE

samplebuffercontaining100 mMTris-HCl,4%SDS,

0.2% bromophenol blue, 20% glycerol and 200 mM

b-mercaptoethanol. The lysate was separated on a

12.5% SDS-PAGE polyacrylamide gel and then

transferred to a nitrocellulose membrane (Bio-Rad

system). In addition, rLPG3 (Rafati et al. unpub-

lished data), rCPB (Rafati et al. 2001) and synthetic

amastin signature (Rafati et al. 2006) were used as a

control. After protein transfer, the membrane was

blocked overnight at 4 xC in TBS (10 mM Tris-HCl,

pH 7.4, 150 mM NaCl) containing 2.5% BSA for

2 h, then incubated overnight with either a rabbit

anti-CPB polyclonal antibody (1 : 500), a mouse anti-

LPG3 polyclonal antibody (1 : 900) or a mouse

anti-amastin polyclonal antibody (1 : 600) in TBS.

The membranes were washed 3 times with TBS,

0.1% Tween 20 and incubated for 1.5 h with either

peroxidase-conjugated anti-rabbit (1 : 4000 Sigma)

or peroxidase-conjugated goat anti-mouse IgG

(1 : 6000, Sigma) as secondary antibodies. Unbound

secondary antibodies were removed by washing

as described above. Diaminobenzidine tetrahydro-

chloride (DAB, Sigma) was used as the substrate to

detect the desired bands of the proteins.

RESULTS

Presence of known virulence factors in the

non-pathogenic Leishmania tarentolae species

First, we looked whether the known virulence factors

of pathogenic Leishmania species CPB, GP63,

LPG3, A2 and Amastin were present in lizard non-

pathogenic L. tarentolae. Total DNA from the

pathogenic parasites L. major and L. infantum and

from the non-pathogenic species L. tarentolae was

digested, transferred to nylon membranes and hy-

bridized with gene-specific probes corresponding to

the coding regions of the above virulence factors.

The results presented in Fig. 1 demonstrate that

the genes for the cysteine peptidase B family, GP63

family, amastin family and LPG3 are present in

L. tarentolae. Interestingly, the band patterns appear

to be similar between the three species especially for

CPB and GP63. The observed multiple banding in

lanes corresponding to CPB and GP63 could be due

to the presence of multiple copies of these genes in

the genomes of these parasites. A similar pattern

in L. tarentolae suggests that these factors are also

present in multiple copies. Detection of the amastin

genes in L. tarentolae required low-stringency con-

ditions for hybridization. Screening of aL. tarentolae

library was performed to seek homologues of the

amastin LmjF24.1270, which is highly conserved

between L. major and L. infantum. Partial screening


led to the cloning of a 900 bp fragment from the

genome of L. tarentolae. Sequencing and BLAST

revealed that this fragment corresponds to the

C-terminal region of the L. major’s Lmj28.1400

orthologue (sequence submitted to GenBank, Ac-

cession no. EU041954). Additional analyses revealed

that the retrieved sequence does not cover the whole

ORF of the gene but a region close to its C-terminal.

Interestingly, among all the virulence factors

examined, the amastigote-specific A2 gene appears

to be absent in L. tarentolae. Low-stringency wash-

ing and prolonged exposure was done to confirm this

result (data not shown).

Genomic structure and organization of known

virulence factors in the non-pathogenic Leishmania

tarentolae species

In this study, a partial sequence of an amastin

homologue and a cysteine protease B gene and the

full sequence of the LPG3 ORF from L. tarentolae

were obtained (sequences submitted to NCBI with

Accession nos EU041954, EU860161 and unpub-

lished data, respectively). A BLAST search with

these sequences throughout the genomes of L. major,

L. infantum and L. braziliensis and subsequent pair-

wise alignment on protein sequences determined

the degree of conservation between these genomes

(Fig. 2). Heat maps shown in Fig. 2 provide a

visual representation of the degree of homology of

amastin, CPB and LPG3 genes between different

Leishmania species. The black colour represents

65.4% similarity (Fig. 2B) (lowest similarity ob-

served in all alignments between L. tarentolae

amastin and L. braziliensis amastin) and the white

colour represents 100% similarity (Fig. 2B). Figure

2A shows that LPG3 is highly conserved among

all the species (lowest 83.5% between L. braziliensis

and L. infantum) in comparison to amastin and

CPB (Fig. 2C). Evaluation of protein homology was

performed using the ClustalW software. Sequence

alignment of amastin orthologues from L. major

(LmjF28.1400), L. infantum (LinJ28.1460), L. bra-

ziliensis (LbrM28_V2_1550) and L. tarentolae

(EU041954) revealed that amastin has the least level

of conservation among the above-mentioned para-

sites (40.07%) with 73.95% sequence similarity only

in the most conserved regions (e.g. transmembrane

helices) (Fig. S1A, see online version). Amastin

proteins are part of a large family of surface proteins

in Leishmania that share a common structure in

L. major and L. infantum. This structure consists

of 4 transmembrane helices and a highly conserved

11-residue sequence between transmembrane helices

1 and 2 that is named the amastin signature (Rochette

et al. 2005). These conserved regions are also pre-

sent in L. tarentolae (Fig. S1A, see online version).

In addition, prediction of transmembrane helices

using the web server TMHMM resulted in the

same transmembrane topology to the other members

of the amastin gene family with the exception of

the last transmembrane region, which is not covered

21226

A2LPG3ama8ama30ama28ama24GP63CPB

LT LM LI LT LM LI LT LM LI LT LM LI LT LM LI LT LM LI LT LM LI LT LM LI

51484973

4268

3530

20271904

15841375

947831

564

Fig. 1. Southern blot analyses of various virulence factors in the human-pathogenic Leishmania major (LM) and

L. infantum (LI) and the non-pathogenic L. tarentolae (LT) species. With the exception of the amastigote-specific

gene A2, the rest of the virulence genes tested are present in the genome of L. tarentolae and show similar digestion

patterns, which suggests a similar copy number and genomic organization.


in the retrieved sequence (Fig. S1B, see online ver-

sion).

Similarly, alignment of the partial sequence of

L. tarentolae CPB (EU860161) shared with L. major

CPB (LmjF08.1030), L. infantum CPB (LinJ08_

V3.0960) and L. braziliensis (LbrM08_V2.0820)

resulted in 59.53% identity and 77.52% similarity

(Fig. S1C, see online version). The maximum level

of sequence similarity was obtained for LPG3 in

which 86.25% similarity between the L. tarentolae,

L. major (LmjF29.0760), L. infantum (LinJ29.0920)

and L. braziliensis (LbrM29_V2_0780) orthologues

(Fig. S1D, see online version) was estimated.

Expression patterns of known virulence factors in the

non-pathogenic L. tarentolae species

As previously reported (Breton et al. 2005), the

L. tarentolae strain used in this study can efficiently

infect human or murine macrophage cell lines as

well as intraperitoneal macrophages freshly isolated

from BALB/c mice, human PBMCs and immature

monocyte-derived dendritic cells, and it can last for

5 days inside macrophages although with a gradual

decrease in infection levels. Therefore, a relative

quantification method (DD CT method) was applied

to evaluate the relative expression ratio of the

Amastin,CPB and LPG3 genes in both promastigote

and amastigote forms of the human-pathogenic

L. major and L. infantum species and of the lizard-

non-pathogenic L. tarentolae. The rRNA45 and

GAPDH genes were utilized as an internal control

for normalizing the expression of the candidate genes.

rRNA45 was reported to be expressed stably be-

tween promastigote and amastigote forms ofL. major

(Akopyants et al. 2004; Ouakad et al. 2007a). Results

on LPG3 expression analysis showed that this gene is

expressed in both life stages of the above-studied

parasites. However, there were some differences

in LPG3 expression levels between amastigotes/

stationary-phase promastigotes of L. major (1.665)

and L. infantum (1.862) versus L. tarentolae (0.926,

Fig. 3). For the CPB gene, a similar pattern of ex-

pression was observed in the 3 species. Indeed, in all

3 species, CPB expression is slightly increased in the

amastigote stage compared to stationary phase pro-

mastigotes (1.234, 1.351 and 1.17 for amastigotes/

stationary promatigotes of L. major, L. infantum and

L. tarentolae, respectively). While amastin of L. in-

fantum shows higher expression in amastigotes than

in stationary phase promastigotes (1.360), inL. major

it appears that this amastin homologue is more

expressed in promastigotes (ratio of amastigotes/

stationary phase promastigotes of 0.57, Fig. 3).

Similar observations regarding amastin expression

were previously reported by Rochette et al. (2005).

Interestingly, it is shown here that stationary phase

promastigotes of L. tarentolae express this amastin

gene homologue in higher levels than amastigotes

A

B

C

Fig. 2. Heat maps illustrating similarity of (A) LPG3,

(B) amastin and (C) CPB among different Leishmania

species. Each square represents one pairwise alignment;

brighter colours represent higher similarity (white for

100%). Black represents 65.4% similarity, which is the

lowest value observed in all alignments (L. tarentolae

amastin vs L. braziliensis amastin). Heat maps were

generated using Mayday (Dietzsch et al. 2006).


(ratio of stationary phase promastigotes/amastigotes

of 1.77).

We confirmed the expression of CPB, Amastin, as

well as LPG3 mRNAs in both developmental stages

of L. tarentolae by real time RT-PCR. As shown in

Table 1, the obtained CT values of the studied genes

(Amastin, CPB and LPG3) in L. tarentolae demon-

strated that the level of expression is close to that in

L. major and L. infantum. The minimum range of

19.89 corresponds to the LPG3 of L. major amasti-

gotes and the maximum of 26.31 is relevant to CPB

of L. infantum amastigotes (Table 1).

Furthermore, we compared whether the proteins

encoded by Amastin, CPB and LPG3 genes were

differentially expressed between L. major, L. infan-

tum and L. tarentolae species. As shown in Fig. 4(A),

the profiles of all Leishmania lysates were similar

according to Coommasie blue staining. Western blot

analysis using specific polyclonal antibodies for each

recombinant protein including rLPG3 (Fig. 4B1),

rCPB (Fig. 4B2) and the Amastin signature in the

form of synthetic peptides (Fig. 4B3), demonstrated

that each of these virulence factors was expressed

at the protein level in L. tarentolae. Although the

reactivity using anti-CPB (Fig. 4B2) showed minor

differences, its presence in the non-pathogenic L. tar-

entolae is indisputable. As elicited from Fig. 4, all 3

proteinswere expressed constitutively in promastigotes

of both the pathogenic (L. major, L. infantum) and

non-pathogenic (L. tarentolae) Leishmania species.

DISCUSSION

Current research has shown that the presence or

absence of certain virulence factors ofLeishmania can

have a dramatic effect on the fate of Leishmania’s

infection. In addition, clinical manifestations of

leishmaniasis can vary depending on the species and

even on strains (Salotra et al. 2006). Several inves-

tigations have been conducted aiming at identifying

and characterizing these genes and their potential

role in the pathogenesis of this disease (Rochette et al.

2005; Alexander et al. 1998; Mottram et al. 2004;

Zhang andMatlashewski, 1997, 2004; Requena et al.

2000). Therefore, determining the role of these fac-

tors during infection provides a precious knowledge

that leads to a better understanding of host-parasite

interactions as well as to the identification of targets

for treatment and vaccination. Recently, compara-

tive transcriptomics together with proteomic analy-

ses of different virulent Leishmania spp. have been

brought into focus (McNicoll et al. 2006; Paape et al.

2008; Rosenzweig et al. 2008; Leifso et al. 2007;

Rochette et al. 2008).

AmaslinCPBLPG3

2.5

2.0

1.5

1.0

Exp

ress

ion

Rat

io

0.5

0.0

L. majo

r

L. majo

r am

astig

ote

L. inf

antum

amas

tigot

e

L. inf

antum

L. tar

entol

ae am

astig

ote

L. tar

entol

ae

statio

nary

pro

mas

tigot

e

statio

nary

pro

mas

tigot

e

statio

nary

pro

mas

tigot

e

Fig. 3. Expression ratios of Amastin, LPG3 and CPB genes in Leishmania tarentolae, L. major and L. infantum

amastigotes and stationary phase promastigotes. Based on these results, LPG3 shows a slight increase in expression in

amastigotes of L. major (amastigotes/promastigotes 1.65) and L. infantum (amastigotes/promastigotes, 1.86) compared

to stationary phase promastigotes ; whereas, in L. tarentolae expression of LPG3 seems to be constitutive (amastigotes/

promastigotes, 0.93). CPB, similarly to all studied species shows a small increase in its expression in amastigote forms

(1.23, 1.35 and 1.17 for amastigotes/promatigotes of L. major, L. infantum and L. tarentolae, respectively). Lastly,

amastin (dark bars) is down regulated in L. major (amastigotes/promastigotes, 0.57) and L. tarentolae amastigotes

(amastigotes/promastigotes, 0.56), while L. infantum shows a slight increase in amastigotes (amastigotes/promastigotes,

1.36). Standard deviation form CT is shown as error bars.


Although Leishmania pathogenicity is most likely

the result of a high complexity with more than one

mechanism involved, it is possible to identify viru-

lence factors by genomic comparisons between

pathogenic and non-pathogenic Leishmania species.

It has been reported that L. tarentolae is likely to be

pathogenic for lizards but the mechanism(s) is un-

known (Dollahon et al. 1973; Orlando et al. 2002).

However, L. tarentolae is unable to cause disease

in mice, even in immunocompromized mice (Breton

et al 2005), and has never been associated with a

Leishmania disease in humans. Here, we investigated

whether known virulence factors of Leishmania

were present and also expressed in the lizard L. tar-

entolae non-pathogenic species to humans. Among

them, we tested the cysteine proteases, GP63, LPG3,

A2 and amastins and found that, with the exception

of A2, all the other proteins were expressed in

L. tarentolae. Using antisense RNA, it was shown

that the L. donovani A2 gene is essential for survival

of amastigotes both in vitro cultured macrophages as

well as in mice (Zhang and Matlashewski, 1997).

Furthermore, using a recombinant A2 protein as

vaccine against L. amazonensis infection in suscep-

tible mice, a robust protection has been obtained

(Coelho et al. 2003). Because A2 protein is truncated

in L. major (Ghedin et al. 1998; Zhang et al. 2003),

it was suggested that A2 might contribute to the

tropism ofL. donovani to visceralization (Zhang et al.

2003). The absence of A2 in L. tarentolae is in-

triguing and it may be associated to some extent with

the loss of pathogenicity of lizard Leishmania in

mammals. This needs to be experimentally vali-

dated, however.

Similar to L. major and L. infantum, L. tarentolae’s

GP63 is a multi-copy gene. The role of GP63 in

pathogenesis has been widely studied (Bordier, 1987;

Medina-Acosta et al. 1993; McMaster et al. 1994;

Yao et al. 2003) although its function in disease

development and also pathogenesis has not been

clearly defined. Chen et al. (2000) have shown that

down regulation ofGP63 expression inL. braziliensis

using antisense RNA strategy led to impaired

internalization of macrophage cells in vitro. On the

contrary, a similar study on the effect of L. major

gp63x/x in disease progression resulted in impaired

macrophage infection, although delayed infectivity

of challenged BALB/cmice was observed (Joshi et al.

2002). On the basis of this finding, it was proposed

that GP63 might contribute to additional pathogenic

pathways such as migration through the extra-

cellular matrix. Interestingly, it has also been shown

(Gregory et al. 2008) that L. tarentolae is able to

perform GP63-dependent cleavages in host proteins

during infection.

In this study we also showed that LPG3,CPBs and

amastins were expressed in L. tarentolae. LPG3 is an

endoplasmic reticulum chaperone and homologue

of human GRP94 (Larreta et al. 2000), which is

believed to be crucial for proper functioning of

LPG synthesis pathway (Descoteaux et al. 2002). In

L. major, it has been shown that mutants lacking this

gene are avirulent (Spath et al. 2000), while in

L. mexicana, it has been demonstrated that LPG is

not essential for macrophage infection as well as

lesion development in mice (Ilg, 2000). It is worth

mentioning that in contrast to the pathogenic

Leishmania, LPG appears to be absent from the

lizard parasite L. tarentolae (Previato et al. 1997).

In this study, we have shown that LPG3 is pref-

erentially expressed in amastigotes of L. major and

L. infantum, which is consistent with similar findings

from L. donovani (Descoteaux et al. 2002). None-

theless, in L. tarentolae, we found that LPG3 gene is

constitutively expressed. Species-specific differences

even between pathogenic Leishmania species have

recently been reported (Rochette et al. 2008). These

differences point to a differential gene regulation

possibly by sequences in the 3kUTR, which generally

are less conserved between species than the protein-

coding sequences (Peacock et al. 2007). Multiple

sequence alignment of the LPG3 protein between

the 4 studied Leishmania species showed a high

level of conservation for the LPG3 orthologues

(86.25%).

Among Leishmania cysteine proteinases (CPA,

CPB and CPC), the role of CPB in pathogenesis has

been extensively highlighted, as L. mexicana de-

ficient in the cpb array genes showed reduced in-

fectivity in experimentally infected BALB/c mice

(Frame et al. 2000; Alexander et al. 1998). It has been

reported that recombinant hybrid proteins encom-

passing CPA and CPB could elicit protection against

L. major in BALB/c mice (Zadeh-Vakili et al. 2004).

Furthermore, the vaccine trial in dogs using cocktails

of CPA and CPB gave satisfactory results (Rafati

et al. 2005), although further study is required.

Alongside these findings, intensive efforts have been

conducted towards identifying the role of CPB in

virulence. Results on L. mexicana revealed that CPB

promotes survival and growth of intracellular para-

sites living in phagolysosomes through inhibition of

Table 1. CT values from two developmental stages

of the three Leishmania species are shown

(According to these results, expression levels of the studiedgenes in L. tarentolae are comparable to those of the otherpathogenic species.)

Species

Obtained CT values

Amastin CPB LPG3

L. major promastigote 22.38 23.31 21.17L. major amastigote 22.98 22.47 19.89L. infantum promastigote 23.96 26.24 22.14L. infantum amastigote 24.02 26.31 21.75L. tarentolae promastigote 25.01 22.73 21.26L. tarentolae amastigote 25.15 22.81 20.69


the NF-kB pathway and degradation of MHC class

II molecules thereby preventing IL-12 production

and eliciting IL-4 expression (Mottram et al. 2004).

However, infection of L. major-resistant mouse

strains with L. mexicana resulted in lesion develop-

ment due to the ability of L. mexicana to cleave a

component of NF-kB, a capacity that seems to be

absent in L. major (Cameron et al. 2004; Buxbaum

et al. 2003). Moreover, these studies demonstrated

that infection of macrophages with wild type or

cpbx/x L. mexicana elicited similar levels of IL-12

production, suggesting that other antigen-presenting

cells (APCs) may be involved in vivo, instead of

macrophages or even modulate other Th1-inducing

cytokines (such as IL-18) (Fukao et al. 2000). Given

the above-discussed functions of CPB, it is sur-

prising that the non-pathogenic L. tarentolae does

possess and express CPB in its genome. Besides,

Southern blot analysis suggested thatCPB is amulti-

copy gene in L. tarentolae, similar to the pathogenic

species. Our analysis showed that CPB is expressed

slightly more in the amastigote stage in all 3 parasitic

species. Such a high level of similarity in CPB ex-

pression, even at the protein level, between patho-

genic and non-pathogenic species is a noticeable

finding that needs additional studying, especially at

the level of protein function. Characterization of this

protein in L. tarentolae will be interesting due to the

differences in protein sequence and possibly struc-

ture in comparison to the L. major and L. infantum

CPB. Heat map analysis and alignments of the

obtained partial sequence of CPB from L. tarentolae

with its equivalent in L. major, L. infantum and

L. braziliensis have predicted 71–77% similarity.

Meanwhile, the maximum sequence similarity of

88.4% was estimated between L. major and L. in-

fantum. Based on this fact, one could conclude that

CPB should have a high degree of heterogeneity over

different Leishmania species, which is compatible

with previous findings on its copy number (Mottram

et al. 2004) and variation in the C-terminal extension

(Hide et al. 2007; Nakhaee et al. 2004; Rafati et al.

2003).

Finally, we analysed the presence and organization

of the amastigote-specific amastin gene family in

L. tarentolae. Amastins are the largest family of de-

velopmentally regulated surface proteins in Leish-

mania (Rochette et al. 2005). Although their role has

not yet been characterized, it is suggested that these

proteins may be involved in the transportation of

ions, metals and nutrients by the internalized para-

sites within the phagolysosome (Rochette et al.

2005). As suggested by Southern blot hybridization,

L. tarentolae also has multiple copies of this gene

family. However, the ongoing sequence of the

L. tarentolae genome indicated a significantly smaller

number of amastins in this non-pathogenic species to

humans compared to the pathogenic Leishmania

(Papadopoulou et al., unpublished observations).

Heat map analysis and sequence alignment of the

L. tarentolae amastin homologue revealed that this

putative amastin protein does possess the 11 amino

acid amastin signature sequence, which is charac-

teristic of all amastin proteins. The results of real-

time PCR showed that amastin is differentially

expressed between amastigotes and stationary-phase

promastigotes. Meanwhile, L. major and L. tar-

entolae express this gene preferentially in stationary

phase promastigotes, whereas in L. infantum this

gene is preferentially expressed in amastigotes as

previously reported (Rochette et al. 2005). Western

blot analysis confirmed amastin protein expression

in L. tarentolae promastigotes, as well. It is worth

mentioning that like LPG3 and CPB, expression

levels of amastin in L. tarentolae are comparable to

those of the pathogenic species. The L. tarentolae

A B

B1

B2

B3

Fig. 4. SDS-PAGE and Western blot analysis of LPG3, CPB and amastin proteins in Leishmania tarentolae,

L. infantum and L. major. (A) Lysates of L. tarentolae (lane 2), L. infantum (Lane 3), L. major (lane 4), rLPG3

(Lane 5), rCPB (Lane 6) and amastin signature (Lane 7) were subjected to electrophoresis on 12.5% SDS-PAGE

and stained with Coomasie blue. (B) Same samples were transferred to a nitrocellulose membrane and incubated with

different polyclonal antibodies including anti-LPG3 (B1), anti-CPB (B2) and anti-amastin (B3) as described in the

Materials and Methods section.


amastin homologue shows 65–72% sequence hom-

ology to the closest orthologues in L. major, L. in-

fantum and L. braziliensis as compared to 89.8%

sequence identity between L. major and L. infantum.

The lower degree of similarity of the L. tarentolae

amastin could affect interactions with other proteins

or substrates and hence modulate amastin’s function.

Amastin’s putative role in pathogenesis has been

recently suggested by DNA microarray data, in-

dicating that amastin genes were differentially ex-

pressed between differentL. donovani strains isolated

from PKDL and VL patients (Salotra et al. 2006).

However, the exact role of amastin in the disease

progression has not yet been identified.

In summary, to our knowledge, this work provides

the first comparative genomic analysis and ex-

pression profiles of important virulence genes, such

as CPB, LPG3 and Amastin in stationary phase

promastigotes and amastigotes between pathogenic

Leishmania spp. and L. tarentolae, the lizard species

non-pathogenic to humans. Our data confirmed that

these virulence genes were expressed and the

proteins produced in L. tarentolae. At this level, we

cannot exclude that differences in protein sequence

or species-specific differential regulation, mainly at

the post-translational level of these virulence factors,

might alter the interaction of these proteins with

other cellular partners, which could result in changes

in the parasite’s virulence. It is also possible that

differences in the thermophysiology between lizard

and mammalian Leishmania species could account

for the loss of virulence of L. tarentolae in mammals.

It is not known what genes are involved in this pro-

cess but it has been reported that some dehydro-

genases and aldolases showed differential expression

between lizard- and human-infecting Leishmania

species (Janovy et al. 1972, Ghosh et al. 1976). This

interesting aspect requires further investigation,

which is outside of the scope of this study. It is also

likely that other, yet uncharacterized virulence fac-

tors, absent or differentially regulated in the lizard

L. tarentolae could account for the loss of patho-

genicity in this species. Whole-genome comparative

analyses between pathogenic and non-pathogenic

Leishmania species will shed more light in that

direction.

The financial support by the Iran Research Council of theRepublic Presidentship is gratefully acknowledged. Inaddition, this project was supported by Pasteur Institute ofIran grant number 282. We wish to thank Mr Alizadeh fortechnical assistance.

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Searching for virulence factors in the non-pathogenic parasite to humans Leishmania tarentolae

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